Regulation of cardiac patterning by the BAF complex subunit Smarcc1a

项目总结 心脏构型是在胚胎内创建具有独特功能的不同区域的基本过程 心。随着心脏的发育，它从一个简单的线性心管转变为一个带有心房的花纹结构 以及室腔和房室管(AVC)的交界处。它们的形成 形态差异伴随着基因表达模式的动态变化：处于 首先在整个心管广泛表达，然后限制在心腔或房室。尽管 AVC和小室模式的功能重要性，我们还没有完全了解其分子机制 这促进了特定基因对任一区域的限制。 为了解决这个悬而未决的问题，我们的实验室对斑马鱼进行了基因筛查，以寻找突变。 这会破坏AVC或心腔的形态。在之前的筛查中，我们发现了一个突变体 AVC和心腔形态发生严重缺陷，导致相对线性的心脏没有 房室交界处形态明显的AVC。此外，变异人的心脏 未能向AVC展示适当的基因表达精细化。位置克隆揭示了 致病突变破坏斑马鱼Smarcc1a基因，编码一种SWI/SNF型依赖于ATP的染色质 与哺乳动物BAF155同源的重塑复合亚单位。这些结果提出了一个新的模型，在这个模型中 含有Smarcc1a的BAF复合体对染色质的重塑细化了基因表达模式 促进AVC和Chamber身份之间的区别。 为了测试这个模型，我将首先确定Smarcc1a何时何地将基因表达限制在AVC。 具体地说，我将研究Smarcc1a对基因表达的区域模式的影响程度， 确定Smarcc1a突变体中首次出现基因表达缺陷的时间，并利用转基因策略 组织特异性抢救Smarcc1a突变体。此外，我将确定作用于下游的效应基因 Smarc1a来驱动心脏模式。使用功能损失、功能增益和上位性分析，我将测试 Smarcc1a是否通过影响其对tbx2b表达的抑制来控制心脏模式。我也会 使用转录转录方法确定更广泛的受Smarcc1a调控的候选基因花名册， 我将测试排名靠前的候选人是否是执行心脏模式的关键效应者。 总而言之，这些研究很可能揭示Smarcc1a在调节区别于其他基因的机制中的新作用。 从相邻心腔的特点来分析AVC的特点。Smarcc1a的这一角色将代表 在心脏模式形成过程中，BAF染色质重塑复合体的功能尚未得到重视。因此，这一点 这项工作有可能产生更广泛的生物医学影响，因为它可能会增强我们对 染色质重塑复合体编码成分的基因突变可能与 先天性心脏病。

PROJECT SUMMARY Cardiac patterning is an essential process that creates distinct regions with unique functions within the embryonic heart. As the heart develops, it transforms from a simple linear heart tube into a patterned structure with atrial and ventricular chambers and the atrioventricular canal (AVC) at their junction. The formation of these morphological distinctions is accompanied by dynamic changes in gene expression patterns: genes that were at first broadly expressed throughout the heart tube become restricted to either the chambers or the AVC. Despite the functional importance of AVC and chamber patterning, we do not fully understand the molecular mechanisms that promote the restriction of specific genes to either region. To address this open question, our laboratory has carried out genetic screens in zebrafish in search of mutations that disrupt the morphology of the AVC or the cardiac chambers. In a previous screen, we identified a mutant with severe defects in AVC and chamber morphogenesis, resulting in a relatively linear heart without a morphologically distinct AVC at the boundary between the atrium and the ventricle. In addition, the mutant heart fails to exhibit the appropriate refinement of gene expression to the AVC. Positional cloning revealed that the causative mutation disrupts the zebrafish smarcc1a gene, encoding a SWI/SNF-type ATP-dependent chromatin remodeling complex subunit homologous to mammalian BAF155. These results suggest a novel model in which chromatin remodeling by Smarcc1a-containing BAF complexes refines the gene expression patterns that promote the distinction between AVC and chamber identities. To test this model, I will first determine when and where Smarcc1a acts to restrict gene expression to the AVC. Specifically, I will examine the extent to which smarcc1a influences regional patterns of gene expression, determine when gene expression defects first emerge in smarcc1a mutants, and utilize transgenic strategies for tissue-specific rescue of smarcc1a mutants. Additionally, I will identify effector genes that act downstream of Smarcc1a to drive cardiac patterning. Using loss-of-function, gain-of-function, and epistasis analysis, I will test whether smarcc1a controls cardiac patterning through its influence on repression of tbx2b expression. I will also use transcriptomic approaches to identify a broader roster of candidate genes that are regulated by smarcc1a, and I will test whether top candidates are key effectors in executing cardiac patterning. Together, these studies are likely to reveal a novel role of smarcc1a in regulating the mechanisms that distinguish the traits of the AVC from the traits of the adjacent cardiac chambers. This role of smarcc1a would represent a previously unappreciated function for BAF chromatin remodeling complexes during cardiac patterning. Thus, this work has the potential to have a broader biomedical impact, as it may enhance our understanding of how mutations in genes encoding components of chromatin remodeling complexes could relate to the origins of congenital heart disease.